Histological tissue specimen treatment

ABSTRACT

A tissue processor for processing tissue samples for histological analysis. The processor comprises two retorts, wax baths, reagent containers, a pump and valve. The vale distributes the reagent from one container to either retort. Separate reagent lines connect the wax baths to the retorts. A method of infiltrating a sample containing a reagent such as a dehydrating reagent like an alcohol, where the infiltrating material is heated to a temperature at or above the boiling point of the reagent, to boil off the reagent when the tissue sample is contacted by the infiltrating material. The pressure in the retort may be reduced to lower the boiling point of the reagent.

FIELD OF THE INVENTION

This invention relates to systems and methods for the processing ofhistological tissue specimens.

BACKGROUND OF THE INVENTION

Histological tissue specimen preparation is a physical process thatinvolves chemical solutions reacting with biological specimens.Typically specimens such as tissue samples from biopsies and autopsiesrequire processing. The end result of such processing is a sample thathas been preserved, and been infiltrated with paraffin. Once the tissuehas been embedded in the paraffin, it is stable and may then besubsequently embedded and then sectioned on a microtome. This processhas typically involved four different sub-procedures:

(a) Fixation

Fixation is a process by means of which cell proteins are stabilised,and the process is normally performed using chemical solutions. A goodfixative is usually a fluid that will neither shrink nor swell thetissue, and more particularly will not dissolve its constituent parts,but will kill bacteria and render enzymes inactive. In addition, thesolution should modify tissue constituents in such a way that theyretain their form when subjected to treatment that would have damagedthem in their initial state. The most commonly used chemical solution isformalin.

(b) Dehydration

Since the ultimate purpose of tissue specimen treatment is to infiltratethe tissue sample in paraffin, and since water and paraffin are notmiscible, the sample must be dehydrated after the fixation step. This isusually achieved by subjecting the tissue sample to increasingconcentrations of alcohols.

(c) Clearing

After dehydration, the tissue sample is still not capable of acceptingparaffin since paraffin and alcohol are not miscible. A chemicalsolution, selected to be miscible with both alcohol and paraffin, isused to clear the alcohol from the sample. The chemical solution mostcommonly used is xylene. Unfortunately, xylene is considered to be toxicalthough most histological processing laboratories use xylene on a dailybasis.

(d) Infiltration

The fourth and final step in the tissue sample treatment is infiltratingthe sample, usually with paraffin wax. In this step the cleared tissuesamples are placed into paraffin heated to a few degrees above itsmelting temperature. Several changes of paraffin may be required toremove the residual xylene so that the tissue is completely infiltratedwith the molten paraffin.

The timing of the fluid change for all the fluids relates to therequirement to effectively displace the previous chemical from thetissue samples. Tissue samples can vary considerably in content andsize, and therefore there may be a large variation in the time requiredto displace the fluid from one sample compared to the time taken todisplace fluid from another. Further, some samples are sandwichedbetween biopsy pads that are porous and absorb significant quantities offluid.

The first attempt at automation of the previously manual method oftissue processing involved placing solutions in a circular arrangementso that samples could be moved from container to container until theyreached the last heated paraffin reservoir. The most well knowninstrument with this type of configuration used in the histology fieldwas the Technicon. One of the major disadvantages of instruments of thistype was that they allowed fumes to escape into the laboratory, thusexposing the laboratory workers to a hazardous environment. To overcomethis problem, the next generation of tissue processing instrumentsincluded a centrally located closed chamber for the tissue samples. Thesolutions necessary for tissue processing were delivered into the closedchamber using suitable valving where the fluids are pumped in and out ofthe chamber in sequence. Normally the chamber would not be opened duringthe process.

As the chamber is closed, and only a single protocol can be run, theprotocol must attempt to cater for the range of tissue samples that maybe included in a single run. This can result in either over processingor under processing of some samples. Given the sealed nature of theretort, tissue samples may not easily be removed or added during aprocessing run.

Another problem is that some samples require urgent processing, whileother samples are not urgent. In the known tissue sample preparationapparatus it has not been possible to stop a current sample run toprocess a sample required urgently, or to employ a protocol that allowsan urgently required sample to be processed with other samples thatrequire longer processing times. Thus, either the urgently requiredsample is run in isolation, or it is put with other samples, increasingthe processing time.

Examples of known automated tissue processing machines will be found inthe patent literature, and typical examples include U.S. Pat. No.4,141,312 Louder, and U.S. Pat. No. 5,049,510 Repasi et al.

The prior art has therefore been unable to deal adequately with ensuringthat a variety of samples can be processed safely and efficiently.

Some systems include heating of wax or tissue samples with microwaves,however microwave systems are difficult to automate, and preferentiallyheat the tissue sample rather than the reagents.

There is a need in the art for a tissue processor that providesautomation to tissue processing.

There is also a need to reduce the use of hazardous chemicals employedin tissue processing.

There is a further need to clear infiltrating material of contaminants.

SUMMARY OF THE INVENTION

In one form, the present invention relates to a tissue processor havingtwo retorts, a controller, and a set of reagent containers fluidlyconnected to the retorts by reagent conduits and a valving arrangement,wherein the valving arrangement directs reagent from the reagentcontainers into either of the retorts, as directed by the controller.

In another form the present invention relates to a method of processinga tissue sample including the steps of:

dehydrating fixed tissue by exposing a tissue sample to a dehydratingsolution;

infiltrating the dehydrated tissue samples by exposing the tissue to aninfiltrating material

wherein the step of removing the dehydrating fluid is accomplished byheating the infiltrating material to a temperature substantially equalto, or above the boiling temperature of the dehydrating solution, andcontacting the tissue sample with the infiltrating material so thatdehydrating solution on the sample boils enabling the infiltratingmaterial to infiltrate the tissue sample.

In a another form the present invention relates to a method of cleaningtissue processor infiltrating material of volatile contaminantsincluding the steps of:

heating the infiltrating material to a cleaning temperature

subjecting the infiltrating material to reduced pressure to lower theboiling point of the volatile contaminants wherein the cleaningtemperature is substantially equal to or above the boiling temperatureof the contaminants at said reduced pressure.

Embodiments of tissue processors of the present invention are describedin greater detail below with reference to the accompanying diagrams.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified schematic block diagram of a first embodiment ofa tissue processor according to the invention showing the basic elementsof a tissue processor;

FIG. 2 is a more comprehensive schematic block diagram of a tissueprocessor showing air and reagent lines;

FIG. 3 shows a perspective view of an embodiment of the tissue processorshown in FIG. 2;

FIG. 4 shows a perspective cut-away view of a retort of the tissueprocessor shown in FIG. 3;

FIG. 5 shows a similar perspective cut-away view of the retort of FIG. 4with cassette baskets in place.

FIG. 6 shows a front view of the retort shown in FIG. 4;

FIG. 7 shows a graph of isopropanol boiling temperature with respect tovacuum pressure.

FIGS. 8 a and 8 b show views of an example of a reagent valve used inthe tissue processor of the present invention;

FIG. 9 shows a rear view of the tissue processor shown in FIG. 3.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In FIG. 1 an example of a general schematic of the processor 10 isshown, indicating major features such as retorts 12 and 14, fourinfiltrating baths 16-22, containers 26, reagent valve 40, manifold 38,and air pump 44. There are three main fluid sub-systems connecting themajor elements, one sub-system being the air lines 30 from pump 44 toinfiltrating baths 16-22 and retorts 12 and 14. A second sub-systembeing infiltrating lines 32 connects infiltrating baths 16-22 to theretorts 16-22. A third sub-system is reagent lines 34 connecting thecontainers 26 to the reagent valve 40 and the retorts 12 and 14. Valvingas shown in FIG. 2 ensures that fluid flows along the lines to thecorrect destination, and FIG. 2 shows a specific embodiment of fluidline connection and valve placement relative to the aforementionedelements. The electrical connections between the controller 25, valves,pump 44 and other elements have been omitted from FIG. 2 for clarity,and are considered standard fittings. Also omitted from FIG. 2 is thenumerous containers 26 and their respective connections to the reagentvalve 40, to provide clarity. The omitted connections are identical tothe connections shown in FIG. 2.

The schematic of FIG. 2 is embodied in the examples shown in FIGS. 3 and9.

With reference to FIGS. 3 and 9, the processor 10 includes controlinterface 24 that employs a graphical user interface to enable a user tooperate the processor by controller 25. In the present embodiment thecontroller 25 is located in cabinet 11, however the interface 24 andcontroller 25 may be located separately, for example as part of astand-alone personal computer. The controller 25 may include a personalcomputer processor such as a Celeron chip by Intel Corporation locatedon an ETX form factor PCB (not shown). The controller may contain anumber of predefined protocols (or steps) for processing tissue, theprotocols being stored in a non-volatile memory such as a hard drive.Protocols may be programmable by the user to implement a number of stepsfor tissue processing, or predefined. Typical protocol parametersinclude which reagents are to be applied to the samples, how long thereagents are to be applied, the temperature at which the reagents areapplied, whether agitation is to take place, and whether ambientpressure in the retort is to be changed.

In FIG. 3, the retort 12 and 14 can be seen in front of infiltratingbaths 16-22. The lids for the retorts 12 and 14 have been removed forclarity, as have the lids for the infiltrating baths. In the presentembodiment each retort 12 and 14 would have a lid (not shown), and eachpair of infiltrating baths would also have a lid 17 and 19 (shown inFIG. 9). The lids may seal with the retorts and baths when in a closedposition. The containers 26 may be located under the retorts 12 and 14so as to be accessible to a user. The controller interface 24 in FIGS. 3and 9 employs a touch screen, however other input and display devicesmay be employed. Also located under the retorts 12 and 14 is a filterunit 52, which typically includes a carbon filter to absorb vapours fromair expelled from the processor 10.

In FIG. 9 the various fluid lines such as reagent lines 34 from reagentcontainers 26 can be seen attached to a reagent valve 40. The reagentvalve 40 may have inputs from all containers 27, and a single output toretorts 12 and 14. A number of air lines can also be seen connectingmanifold 38 to the reagent bottles 26. The connections between variouselements in FIG. 9 are shown schematically in FIG. 2.

One embodiment of retort 12 is shown in FIGS. 4-6, including areceptacle 13 for receiving baskets 62 containing tissue samples. Thereceptacle has a working capacity of 5.5 liters, however it may notnecessarily be completely filled during each step of a protocol. Whenlocated in the processor, the retort may be rotated 10 degrees forwardtowards the front of the processor 10. This allows easier access to thebaskets, as well as providing a drainage point which is lowermost in thereceptacle 13, minimising residuals remaining in the retort 12 afterdraining.

Sensors 52 are used to detect the level of fluid within the retort 12,so that the controller 25 can ascertain when to turn the pump 44 on oroff, or open and close the appropriate valves, as described below. InFIG. 6, the placement of the three sensors 52 can be seen. The lowermostsensor detects when the level of liquid, for example reagent orinfiltrating fluid, is above a minimum level. The minimum level mayrepresent a partially filled receptacle which is desirable whenoperating in economy mode. This is desirable when two or less basketsare to be processed at once, whereupon only approximately 3.8 liters offluid are required to cover the baskets and samples contained therein.As the baskets may be various sizes, the level of the lowermost sensorand therefore fill volume for economy mode can vary in differentembodiments of the retort 12. The middle sensor 52 detects when thelevel of liquid typically covers three baskets, which is a normal fullload. The top sensor 52 detects an overfill situation. In thisparticular embodiment the sensors are optically based relying on achange in refractive index when liquid comes into contact with a prism(not shown) of the sensor. Each basket may hold approximately 100samples either in individual cassettes or placed directly into thebasket. Thus a full load for the embodiment of the retort 12 shown inFIGS. 4-6 is approximately 300 samples. The retorts may be made largeror smaller depending on requirements.

Also shown in FIG. 6 is temperature sensor 53 which is mounted directlyto the retort 12, and temperature sensor 54 which is mounted to aheating mat 55. The retort 12 is heated to ensure correct reagent, orinfiltrating fluid temperature. Placing a temperature sensor directly onthe retort 12 allows the fluid temperature within to be measured moreaccurately than by measuring the temperature of the heating mat,especially where the fluid used may have low thermal conductivity. Thetemperature of the heating mat may then be kept at a maximum while thetemperature of the retort 12 is below the maximum, providing more rapidheating than if only one temperature sensor was employed.

Port 56 shown in FIG. 6 allows connection of an air line 30 to theretort 12. Retort manifold 57 also allows connection of infiltratingline 30 and reagent line 34 through a common entry point (not shown) atthe bottom of the receptacle 13. In FIG. 2, retort manifold 57incorporates valves ret1-vrgt and ret1-vwax, and is located at the frontof the processor 10 so that the lean angle of 10 degrees of the retortcauses all fluid to drain towards the common entry point.

In FIGS. 4 and 5, the interior of the receptacle 13 is shown, includingagitator 70. Agitator 70 is magnetically coupled to an electric motor58, and may be driven at a number of speeds dictated by controller 25.The baskets 62 each contain up to 100 tissue samples. The baskets 62 aresupported clear of the agitator on posts 59 shown in FIG. 4.

In the present example, retort 12 and 14 are of identical construction,size and operation, however one retort may be larger or more volumousthan the other. Connections to and from retort 12 are duplicated onretort 14.

In FIG. 2, pressure relief valves 48 are shown in fluid communicationwith air lines 30, retorts 12 and 14, and the infiltrating baths. Anyoverpressure in these lines will result in excess air being vented towaste through the manifold and filter 47.

A list of valve functions is as follows:

Valves ret1-vwst and ret2-vwst connects retorts 12 and 14 to wastecontainer 72, when a waste cycle is required. Only one retort will beemptied at once and therefore these valves only open one at a time. Inanother embodiment, the valves ret1-vwst and ret2-vwst may be omitted,and waste container 72 may be directly connected to the reagent valve40. To drain to waste are a reagent, the reagent valve 40 connects tothe reagent line 34 connected to the waste container 72, and the valveon the retort is opened to drain reagent directly to the waste container72.

Valves ret1-vrgt and ret2-vrgt allow reagent flow into and out of theirrespective retorts during filling and draining of the retort. Whendraining a retort, these valves are open so that reagent may flow backdown the reagent line and back into the same reagent container 26 fromwhence it came. It can be seen that air valves ret1-vfls and ret2vflsconnect to the reagent lines 34 below the ret1vrgt and ret2-vrgt valves.These air valves are used to purge excess reagent from the reagent linesafter filling one retort. This is desirable as using reduced pressure todraw fluid into a retort reduces fluid pressure along the whole reagentline 34, and therefore when pressure is restored to the reagent line 34some reagent may travel up the line of the retort that was not filled.Opening these valves, or opening the valves and pumping air down the airlines into the reagent lines clears excess reagent, preventing orreducing cross contamination.

Valves ret1-vwax and ret2-vwax connect the retorts to the infiltratingbaths, via infiltrating lines 32 and valves wb1-vwx to wb4-vwx. Valvesret1-vwax opens when infiltrating fluid is to enter or drain from retort12, and wb1-vwx to wb4-vwx open one at a time depending on where theinfiltrating fluid is being sourced. The infiltrating line 32 betweenthe infiltrating baths and retorts is heated to ensure that theinfiltrating material does not harden in the lines.

Valves ret1-vair and ret2-vair are used to control air from the air pumpto the retorts. Air may be supplied either at a positive pressure toambient, or withdrawn from the retorts so that pressure inside one orboth retorts is below ambient pressure. These valves determine whichretort is in fluid connection with the air pump. Also air-vprs must beopen to allow communication between the pump and the valves, otherwiseair is directed toward wax-air valve, connected to the infiltratingbaths.

The reagent valve 40 is shown in FIGS. 8 a and 8 b, and includesconnections between the reagent lines 34 from the reagent containers 26on the input side, and outlet 35, which is fluidly connected to theretorts 12 and 14. The reagent valve 40 selects which reagent containerwill be in fluid communication with the reagent line connected to theretorts. In the present embodiment, the reagent lines 34 from thereagent containers 26 are arranged in a circle attached to the reagentvalve housing 37. In the present embodiment, the reagent valve 40 is inthe form of a rotary valve, having two ceramic discs 39 and 41, disc 39having a single aperture 43 a aligned with aperture 43 b to form aconduit for reagent. The discs are mounted coaxially and adjacent eachother and rotate together according to the position dictated by thecontroller 25. Disc 45 has an aperture for each reagent line 34,although in FIG. 8 b only one aperture is in the plane of the crosssection. The rotating discs 39 and 41 rotate with respect to disc 45,driven by stepper motor 49 such that the apertures align to provide aflow path from the outlet 35 (and therefore one retort) to a reagentcontainer 26. In order to assist with sealing between the discs 39, 41and 45, a plate 51 applied pressure to the discs. In this way anyreagent line 34 and therefore any reagent container can be selected bythe controller 25 to be in fluid communication with one of the retorts12 or 14. This type of valve has a small internal volume and thereforeminimises cross contamination. Further, the reagents are drained backinto the reagent containers after each step and therefore little reagentremains to contaminate the subsequent reagent. It should be noted thatthe infiltrating fluid does not pass through the reagent valve. Thisseparation of fluid flows prevents the reagent valve from clogging andreduces the amount of cleaning of the valve.

In use, the tissue samples to be processed are typically placed intocassettes (not shown) for placement into a basket 62. Generally, tissuesamples expected to have similar processing times and to be exposed tothe same processing protocol are placed together in the same basket 62.The basket 62 containing the tissue samples is then placed into one ofthe retorts 12 or 14, and the lid closed, forming a sealed enclosure. Anoperator may then enter data into the control interface 24 to instructthe controller 25 of the protocol to be followed. The protocol may beprogrammed step by step, for example indicating the time, temperature,pressure, agitation and reagent for each step, or a pre-programmedprotocol encompassing all steps may be selected.

The first step in a protocol, once the lid of the retort is secured, maybe to fill the chosen retort (in this example retort 12 is chosen) witha fixing solution. A typical fixing solution is formalin, which may beheld in one or more reagent containers. In order to fill the retort 12with fixing solution, the pump 44 is switched on and valves open the airlines from the retort 12 to the inlet side of the pump, pumping air fromthe retort 12 chamber. The reagent valve is set to a position thatfluidly connects the reagent line of the retort 12 to the specifiedreagent container for formalin. Other valves are opened along thereagent lines from the retort 12 to the reagent valve 40. The reducedpressure in the retort 12 is sufficient to draw fluid out of the reagentcontainer, through the reagent valve into the reagent lines 34 and intothe retort 12. The retort is heated by heater pads to a predeterminedtemperature selected and controlled by the controller. Sensors 53 and 54may be used to control the temperature of the retort, and therefore thetissue and any reagent contained therein. One or more sensors 52 in theretort as shown in FIGS. 4 and 6, may be used to detect the reagentlevel. When the reagent level in the retort is sufficient, typically tocover the baskets 62 as seen in FIG. 5, the pump may be turned off orotherwise disengaged from the retort 12, for example by closing valveret1-vrgt shown in FIG. 2.

After a length of time determined by the controller 25 (typically asprogrammed by the user), the reagent may be removed from the retort 12.This is accomplished by opening valve ret1-vair in the air line 30 andopening valve ret1-vrgt in the reagent line 34. Reagent will then drainfrom the retort 12 back into the reagent container from which it came,or back into a different reagent container, or to waste, according tothe position of the reagent valve 40 determined by the programmedprotocol. To assist in draining, the retort 12 may be positivelypressurised by air from the pump 44, supplied along the air lines 30. Inthe present embodiment the reagent drains back to its originatingcontainer. If the reagent is contaminated, or has been used for thepredetermined number of samples or washes, then it is drained to wasteusing a separate waste cycle.

During the retort filling with reagent from a reagent container, the airpumped from the retort 12 flows down an air line 30, some of which flowsback though manifold 38 and into the reagent container, recirculatingsome of the air from the retort 12. Excess air pumped from the retort 12will flow out through a condensing mechanism such as a condensing coil51, and/or a carbon filter 47, both of which are designed to removevolatile organic or other compounds from the air before it reaches theatmosphere. The processor 10 may have an outlet connection that allowsthe filtered air to be vented or further filtered by apparatus externalto the processor 10.

The second step in tissue processing may be the dehydration step. Themethodology employed to draw dehydrating reagent into the retort 12 maybe the same as described above, as the dehydrating reagent will bestored in a reagent container 27. The dehydrating fluid may contain afluid such as an alcohol, for example ethanol. The dehydrating fluid mayalso contain some water, either intentionally added, or, where thedehydrating fluid has been re-used, water removed from previous samples.There may be a number of steps of the protocol where dehydrating fluidis applied to the sample in the retort, and at each step a differentdehydrating fluid may be used. For example, a fluid may be used that hasless water than a previous fluid, to draw out more moisture from thesample at each wash. The dehydrating fluid may additionally oralternatively contain isopropanol. Later washes with isopropanol provideproperties that may be advantageous, as will be described below. Furtheradditives commonly used in tissue processor dehydration fluids may beused, as the present embodiments are intended to be compatible withknown dehydration fluids.

On a final wash with dehydrating fluid, the fluid is drained completelyfrom the retort. This is accomplished by opening valves from the airpump as well as pumping air into the reagent lines to clear the reagent.A vapour flush may be employed where the pump flushes fresh air into theretort to clear any vapour from the reagent, such as a dehydratingfluid. Significant vapour may be present as the dehydrating fluid mayhave high partial pressure at the retort operating temperature. Afterthe dehydrating step, a drying step may be employed, where the retort isheated by the heating mats 55, while air is pumped through the chamberby the air lines 30. This removes excess dehydrating fluid. The dryingstep may take several minutes or more, and the retort may be heated to85 degrees Celsius, depending on the dehydrating fluid chosen and thesensitivity of the tissue samples to heat.

Another step in tissue processing is infiltrating of the samples. Thisis typically accomplished by an infiltrating material such as a paraffinwax. The wax is held in the infiltrating baths 16-22, which are heatedto the desired temperature above the waxes melting temperature, which istypically 54 degrees Celsius. Wax pellets are typically added to aninfiltrating bath, which heats the pellets until they melt and achieve asuitable temperature. Alternatively, pre-molten wax may be addeddirectly to the baths. The wax is held at the elevated temperature,typically 65 degrees Celsius, until required. The present embodimentshows four infiltrating baths, however there may be more or lessdepending on retort and infiltrating bath volume. The infiltrating lines32 run from the infiltrating baths 16-22 to both retorts 12 and 14, andinclude valves such as ret1-vwax and ret2-vwax, that allow one, some, orall baths to be fluidly connected to one of the retorts. The arrangementof the baths, valves, and infiltrating material lines enables samples inone retort to be washed with up to four different infiltratingmaterials. Further, the infiltrating material can be heated in one ormore baths while the processor 10 is in operation and drawinginfiltrating material from the remainder of the baths.

During the infiltrating stage, the wax is drawn into the retort 12 byopening the valve between the retort and appropriate infiltrating bath,such as ret1-vfls, then reducing the pressure in the retort using thepump 44 and opening valves air-vprs and ret1-vair. The reduced pressurein the retort draws the wax into the retort 12. Typically the pressuremay be −20 to −80 kpa gauge, however a wide variety of pressures may beused, and these are user programmable via the controller. The wax may beheated to a temperature above or approximately the same as the boilingtemperature of the dehydrating fluid used in the last or last fewwashes. If an isopropanol is used, the boiling temperature will beapproximately 82 degrees Celsius at atmospheric pressure. Ethanoltypically boils at 78 degrees Celsius. After the retort has been drainedof dehydrating fluid, some fluid remains on or absorbed by the tissuesamples. The tissue samples may then be subjected to a drying stage asdescribed above to remove further dehydrating fluid, and the retortflushed with clean air. Wax is then drawn into the retort. Upon contactwith the heated wax, the remaining dehydrating fluid is evaporated orboiled off the tissue samples, and the wax replaces the dehydratingfluid, thus infiltrating the samples. The pump may continue to draw offair or vapour from the retort to reduce the pressure in the retort,which will reduce the evaporation temperature of the dehydration fluid.As an example, the pressure in the retort may be reduced by 50 kpagauge, resulting in a boiling temperature of approximately 52 degreesCelsius for the isopropanol. A graph of boiling temperature compared tovacuum pressure is shown in FIG. 7. Reducing temperatures of the waxcontacting the tissue samples may provide an advantage, for examplewhere certain types of tissues do not perform well when exposed to hightemperatures. Typically the paraffin wax used (Paraplast+ from OxfordLaboratories) melts at about 54 degrees Celsius. Other infiltratingmaterials may be used including resins used in histological processesfor infiltrating tissue samples. In the present example the alcohol usedat the last stage, isopropanol, is not substantially miscible withparaffin wax. The means that infiltrating fluid is unlikely to penetratethe tissue sample if the previous fluid in the retort was immisciblewith the infiltrating fluid. Boiling the volatile dehydrating materialoff therefore enables the omission of a step whereby an intermediaryfluid such as xylene, which is miscible in alcohol and paraffin wax, isrequired: Xylene has undesirable properties in a laboratory. However,xylene will also evaporate when exposed to temperatures around 80degrees, especially when the pressure inside the retort has been loweredby applying a vacuum as described herein. Thus the present exampleenables the tissue samples to be used without a xylene wash cycle, butalso may be used with fluids such as xylene. There are advantages in notusing xylene, including that xylene is miscible in wax, and thereforecan be absorbed into the wax as a contaminant. However in some instancesit is desirable to use xylene, for example when the tissue requiresclearing and the dehydrating fluid such as isopropanol is deemed to beinsufficient. Further, xylene may be used after a processing cycle toclean excess wax from the retort, and therefore xylene may be present inthe processor.

It is possible to clean the infiltrating fluid of some of the volatilecontaminants, such as the dehydrating fluid, clearing fluids such asxylene, by holding the wax in the bath and reducing the pressure in thebath. This clean cycle is done with the bath lid closed, whereupon thereduced pressure and holding the infiltrating material at an elevatedtemperature such as between 60 degrees and 100 degrees Celsius. In oneembodiment the temperature may be held between 65 degrees and 85 degreesCelsius. By volatile material, it is meant that at the temperaturesmentioned herein, and/or at reduced pressures, the material will boil orevaporate.

The vapour pressure of the dehydration fluid within the air in thecontainer may also be reduced, for example by venting the air in theretort, either while maintaining a low pressure or cycling throughpressure ranges. The infiltrating fluid may be held in the bath at anelevated temperature for several hours to clean away contaminants.

The use of two retorts allows two sets of baskets to be processed eithersimultaneously or with an overlap. Thus one retort can be loaded and aprotocol begun while the other retort is mid-way through the same or adifferent protocol. This provides additional flexibility in theprocessor.

The tissue samples referred to in may be human or animal tissue samples,or samples from plant material.

An example protocol for tissue samples, such as a 3 mm punch humanbiopsy sample, will now be described:

Time Temp Step Reagent (min) (c) Retort Pressure Agitation 1 Formalin 560 ambient yes 2 50/50 ethanol water 25 60 ambient yes 3 80/20 ethanolwater 35 60 ambient yes 4 Isopropanol 30 60 ambient yes 5 Paraffin Wax40 85 Vacuum yes 6 Paraffin Wax 5 85 Vacuum yes total time 140

Another protocol is as follows

Time Temp Retort Step Reagent (min) (c) Pressure Agitation 1 formalin 6040 ambient yes 2 80% ethanol 45 40 ambient yes 3 90% ethanol 45 40ambient yes 4 100% ethanol 60 40 ambient yes 5 100% ethanol 60 40ambient yes 6 100% ethanol 60 40 ambient yes 7 100% ethanol 60 40ambient yes 8 Isopar or d-limonene 60 40 ambient yes 9 Isopar ord-limonene 75 40 ambient yes 10 Isopar or d-limonene 75 40 ambient yes11 Paraplast 70 60 Vacuum yes 12 Paraplast 60 60 Vacuum yes 12 Paraplast60 60 Vacuum yes total processing time 790

From the above it can be seen that xylene is not required in thisprotocol, and that the protocol has few steps, saving time.

In one embodiment a contamination detector 68 may be placed in thereagent line 34 to detect the presence of contaminants in the reagents.

To drain the retort 12, the pump may increase pressure in the retort 12by pumping air along the same air lines 34 as used to draw reagent intothe retort 12. Waste reagent may be drained into a reagent container, orbe expelled to waste port 72. Infiltrating fluid may also be drainedfrom the retort 12 to waste 70 by this method, and similarlyinfiltrating fluid may be drained from the baths using positivepressure.

In the above examples the dehydrating fluid is immiscible with theinfiltrating material. However, the above process offers advantages evenif a clearing cycle is used, where the clearing fluid is miscible withthe dehydrating fluid and the infiltrating material. Further, additivesmay be used to increase the clearing properties of the dehydratingmaterial, as well as increasing the miscibility of the fluids in thedehydrating and infiltrating steps.

While raising the temperature of the infiltrating fluid above theboiling temperature of the dehydrating reagent (or clearing reagent)will result in faster removal of the reagent, reagent will still beremoved at or around the boiling temperature provided the partialpressure in the retort is lower than the partial pressure of the reagentat the given temperature. This can be accomplished by reducing thepressure in the retort, then allowing some fresh in into the retort.Bringing fresh air into the retort while removing air laden with vapourwill reduce the partial pressure of reagent in the air in the retortthus promoting more evaporation of the reagent. If the reagent ismiscible with the infiltrating fluid it may not be necessary to removeall the reagent to obtain infiltration. However, if the samples canwithstand the temperature it is preferable to raise the temperature ofthe infiltrating fluid within the retort to a temperature above theboiling temperature of the reagent for the given pressure. A temperatureabout the boiling temperature of a reagent for a given pressure may betypically a few degrees, such as 5 degrees Celsius, of the boilingtemperature.

Other dehydrating fluids are contemplated as being able to be used withthe present apparatus, such as:

methanol

butanol

ethylene glycol

propylene glycol

Industrial methylated spirits

Denatured alcohol (including alcohol denatured with kerosene, benzene orbrucine)

Reagent grade alcohols

acetone

and combinations thereof, however the above list is merelyrepresentative and is not intended to encompass an exhaustive list ofreagents useful in the processor described herein.

Clearing reagents such as di-pentene, D-limonene, 1,1,1,trichloroethane, toluene, and dioxane are also contemplated, and againthis list is meant to be indicative of the types of reagents that may beused, rather than an exhaustive list. The reagents above, and otherreagents suitable for histological processes such as dehydrating,clearing or a combination thereof, may be used in the present apparatuswith the step of evaporating the reagent from the sample using heatingof the infiltrating fluid, provided the reagents evaporate withoutleaving a residue. While reagents such as butanol have a boiling pointof approximately 118 degrees Celsius at atmospheric pressure, theboiling point drops dramatically with a reduction in ambient pressure.While it is believed preferable to not heat most tissues above 85degrees Celsius, some types of well fixed tissue will survive thistemperature without damage, and therefore higher temperatures may beused, increasing the range of reagents useful in the abovementionedprocesses. Accordingly, the upper temperature which may be used isdependent on the tissue, and therefore in well fixed tissue,temperatures may exceed 100 degrees Celsius. Reducing pressure in theretort will assist in reducing temperatures in the retort by reducingthe boiling point of reagents.

Infiltrating materials such as resins and other fluids used inhistological tissue processing are also contemplated in the aboveexamples, and the present invention is not intended to be limited to theinfiltrating materials mentioned herein. It is also contemplated thatinfiltrating material may be a mixture of substances, such as mineraloils and paraffin wax.

1. A tissue processor having a retort with a first temperature sensormounted to the body of the retort, and a second temperature sensormounted to a heating device of the retort, wherein the tissue processorcomprises a controller that executes a method of heating the retort to adesired temperature the method comprising: measuring the temperature ofthe retort utilizing said first temperature sensor; heating the retortusing said heating device in contact with the retort; measuring thetemperature of said heating device in contact with the retort utilizingsaid second temperature sensor; and keeping the heating device at atemperature below its maximum operating temperature but above thedesired retort temperature until the retort temperature reaches thedesired temperature.